Polyadenylation mRNA Regulation
Overview Polyadenylation of messenger RNA (mRNA) is one of the key steps involved in eukaryotic pre-mRNA processing. The other steps include capping, splicing, and cleavage. Only processed or mature mRNA can be translated. Hence, polyadenylation is very important for gene expression regulation in eukaryotes. Polyadenylation occurs right after transcription of a gene terminates. This is a two-step process that occurs in the maturation of all eukaryotic mRNAs. The first step is endonucleolytic cleavage of pre-mRNA at the 3’-end. The second is polymerization of the poly-A tail to the 3’ end. The poly-A tail is formed when a set of proteins cleaves off the 3’ end of pre-mRNA transcript and then produces the poly-A tail that will be added there. The poly-A tail is essential for stability of mRNA, exit of mRNA from nucleus, and efficient translation. There are several places the poly-A tail could be inserted, which means that alternative polyadenylation can occur because more than one mRNA transcript can be produced from a single gene. Alternative polyadenylation (APA) results in mRNA sequences with differing 3’ ends. When polyadenylation is not well regulated, many diseases such as cancers can result due to variance in gene expression. The length of the poly-A tail is also important because tails that’re too short are subject to enzymatic degradation. Many factors are involved in the selection of a 3’-end processing site among the multiple potential poly(A) sites a gene has. Molecular Mechanism of mRNA 3'-End Processing Constitutive 3'-End Processing Many factors contribute to regulation of the poly-A site. They’re responsible for recognition, cleavage, and polyadenylation in pre-mRNA. The poly-A signal is usually an AAUAAA hexamer or a sequence similar to it. Cis-acting elements and trans-acting proteins regulate the recognition and action mechanisms of polyadenylation. Mistakes in regulation lead to changes in gene expression and cause diseases. Alternative polyadenylation (APA) at the 3’ UTR results in production of multiple mRNAs in most genes. The variable lengths at the 3’ UTRs serve as a recognition site for various regulatory mechanisms, which controls the ultimate product of the mRNA transcript downstream, thus changing the gene expression profile. Polyadenylation and APA are both important for many disorders because irregularities in the 3’-end processing mechanisms have lead to many oncological, immunological, neurological, and hematological disorders. The molecular mechanisms underlying polyadenylation regulation of gene expression have not been fully understood yet. A possible mechanism is promotion of polyadenylation at proximal poly(A) sites due to strong transcriptional activators, which recruit a component of the transcription elongation complex (PAF1c) to the promoter as well as the 3’-end processing complex. Frequently transcribed genes are usually processed at proximal poly(A) sites while those that are less frequently transcribed are processed at distal poly(A) sites. It’s also possible that nucleosome presence in a gene can regulate Pol II elongation rate thus affecting poly(A) site choice. Predicting the implications of different alternative poly(A) sites on gene expression has been a challenging task. Site selection could depend on sequence motifs near the cleavage site of mRNA and on sequence motifs, which bind to secondary factors. In addition, RNA secondary structure and chromatin marks contribute to site selection. Elements & Proteins A system of cis-acting RNA sequence elements and trans-acting proteins regulate mRNA 3’-end processing. They help determine the qualitative and quantitative changes in gene expression. The specific mechanisms are currently being studied, but below are some of the newest updates. Cis-Acting RNA Sequence Elements The arrangement of these elements determines the efficacy of the poly-A site. The most important one is a hexamer, which either has the nucleotide sequence AAUAAA or AUUAAA in 70% of human RNAs. In the rest of RNAs, the sequences are UAUAAA, AACAAA, or ACUAAA. Proteins Involved in 3' End Processing Cleavage of mRNA in the nucleus and subsequent polyadenylation requires four multi-subunit complexes, which are CPSF, CstF, CFIm and CFIIm. These four are called the core 3’-end processing factors. Other factors that're required include the single subunit nuclear poly(A)-polymerase PAP and the nuclear poly(A)-binding protein (PABPN). In eukaryotes, nuclear poly(A) polymerases add a poly(A) tail with a length of up to 250 nucleotides to the 3’ UTR (untranslated region). The length of the tail is determined by poly(A)-binding protein 1 (PABPN1). PAPN1 is replaced by PABPC, a cytoplasmic poly(A)-binding protein, when the mRNA leaves the nucleus. In Xenopus oocytes, ''nucleic pre-mRNA polyadenylation, cytoplasmic deadenylation, and readenylation of mature mRNAs have been observed. Regulated 3'-End Processing Quantity of gene expression can be changed by the amount of polyadenylated mRNA and protein produced as a result. The focus of current research is on mechanisms that regulate polyadenylation efficiency and alternative polyadenylation signal (PAS) usage. In quantitative regulation, formation of specific mRNPs can either stimulate or inhibit gene expression in 3’-end processing (see Figure 2). Qualitatively, mRNAs with multiple polyadenylation signals undergoes alternative polyadenylation (APA). In the case of Figure 2, both PASs (PAS 1 and PAS 2a) are in the 3’ UTR, which result in multiple mRNA variants that produce the same protein (in this case protein 1) but vary in 3’ UTR lengths (mRNA 1, mRNA 2a). One includes miRNA and RBP binding sites while the other doesn’t. If there is a PAS in the coding region such as the case with PAS 2b in the figure, APA will produce two mRNA isoforms (mRNA 2a and mRNA 2b) with different C-terminal coding regions. This leads to translation of two different proteins (protein 1 and protein 2). Proteins Regulating 3'-End Processing RNA-binding protein (RBP) and proteins that assemble at the cis-acting regulatory RNA elements in a transcript primarily regulate mRNA 3’-end sequencing. Some of these proteins include TFIID (transcription factor II D), tumor suppressor p53, and heat shock factor protein HSF1. Nuclear poly(A)-binding protein 1 (PAPBN1) levels have been recently show to be affected by PAS usage. PAPBN1 binds to poly(A) sites. More upstream poly(A) site usage occurs when there's a PAPBN1 deficiency. The exact mechanism behind how PAPBN1 affects PAS usage remains unclear. Hsp70 Gene Regulation by Polyadenylation Heat shock triggers the expression of heat shock proteins (Hsp), which provide cytoprotection. Hsp protects cells against harmful agents. This includes protecting the heart from ischemia or reperfusion (I/R) injury. The Hsp70 family of proteins is one of the most researched heat shock proteins and has been shown to be activated by cardiac stimuli. In this study, the Hsp70.3 gene is shown to undergo post-transcriptional regulation through alternative polyadenylation of its mRNA transcript. A heat shock or ischemic stimulus causes shortening of the 3’-UTR through alternative polyadenylation of the Hsp70.3 mRNA transcript. This results in the elimination of the binding site for miR-378 from the final mature mRNA transcript. miR378 has suppressive properties and functions. Removal of the suppression allows for more stable mRNA transcript and elevated translational efficiency, which results in increased protein expression. This experiment demonstrates how alternative polyadenylation in the heat shock stimulated Hsp 70.3 gene controlled its post-transcriptional protein expression. The increased expression of Hsp70.3 is critical for cardioprotection against acute I/R. Tranter et al. concluded that APA plays a key role in one of several regulatory steps that control Hsp70.3 gene expression. (6) References 1. Wikipedia article: Polyadenylation. Date accessed: Dec. 5, 2014. 2. Elkon, Ran, Alejandro P. Ugalde, and Reuven Agami. "Alternative Cleavage and Polyadenylation: Extent, Regulation and Function." ''Nature Reviews Genetics 14.7 (2013): 496-506. 18 June 2013. Web. 7 Dec. 2014. 3. Hollerer, Ina, Kerstin Grund, Matthias W. Hentze, and Andreas E. Kulozik. "MRNA 3′end Processing: A Tale of the Tail Reaches the Clinic."EMBO Molecular Medicine 6.1 (2013): 16-26. 9 Oct. 2013. Web. 7 Dec. 2014. PMID: 24408965. 4. Gruber, Andreas R., Georges Martin, Walter Keller, and Mihaela Zavolan. "Means to an End: Mechanisms of Alternative Polyadenylation of Messenger RNA Precursors." Wiley Interdisciplinary Reviews: RNA 5.2 (2014): 183-96. 14 Nov. 2013. Web. 7 Dec. 2014. PMID: 24243805. 5. Curinha , A. et al. "Implications of Polyadenylation in Health and Disease." Nucleus 5.6 (2014): n. pag. Web. 7 Dec. 2014. PMID: 25191751. 6. Tranter, M., R. N. Helsley, W. R. Paulding, M. Mcguinness, C. Brokamp, L. Haar, Y. Liu, X. Ren, and W. K. Jones. "Coordinated Post-transcriptional Regulation of Hsp70.3 Gene Expression by MicroRNA and Alternative Polyadenylation." Journal of Biological Chemistry 286.34 (2011): 29828-9837. Web. 7 Dec. 2014. PMID: 21757701.